Method for re-sensitizing vancomycin resistant bacteria which selectively cleave a cell wall depsipeptide

ABSTRACT

The present invention relates a method for re-sensitizing vancomycin resistant Gram-positive bacteria in which resistance results from the conversion of an amide bond to an ester bond in the cell wall peptide precursors of the bacteria which comprises using an antibacterial amount of vancomycin or a homolog of vancomycin and an amount of an agent effective to selectively cleave the ester bond so as to thereby re-sensitize vancomycin resistant bacteria.

The invention disclosed herein was made with Government support underNIH Grant No. 5-R01-HL-25634-18 from the National Institutes of Health.Accordingly, the U.S. Government has certain rights in this invention.

Throughout this application, various references are identified bycitations or a number in parenthesis, in which case their full citationsappear on the pages following the Detailed Description immediatelypreceding the claims. Disclosure of these references in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention relates to a method for re-sensitizing vancomycinresistant Gram-positive bacteria in which resistance results from theconversion of an amide bond to an ester bond in the cell wall peptideprecursors of the bacteria which comprises using an antibacterial amountof vancomycin or a homolog of vancomycin and an amount of an agenteffective to selectively cleave the ester bond so as to therebyre-sensitize vancomycin resistant bacteria.

BACKGROUND OF THE INVENTION

The introduction of the first antimicrobial agents allowed physiciansand patients to manage infectious diseases effectively. Unfortunately,even before penicillin was introduced commercially, researchers hadidentified the first resistant Staphylococcus aureus (1). Since then,many new antimicrobials have been developed to combat the emergence ofresistance. These advances increased the confidence that infectiousdiseases are non-fatal and still manageable. Nevertheless, with themedical progresses that allow people to live longer and the boost ofvarious debilitating immune conditions (AIDS, cancer, organtransplants), a new human population emerged as being at great risk ofinfectious diseases caused in majority by two common nosocomialpathogens, Staphylococcus and Enterococcus species (2). The incidence ofstaphylococcal strains resistant to virtually all the antimicrobialagents except vancomycin has increased drastically during the lastdecade. Additionally, enterococcal infections are becoming increasinglyresistant even to vancomycin, raising the alarming possibility thatresistance genes will eventually be transmitted to staphylococci (3-5).One of the greatest concerns with vancomycin resistant enterococci (VRE)is that the resistance to vancomycin will be picked up by Staphylococcusaureus through genetic recombination. Many forms of S. aureus havealready become resistant to methicillin and can now only be treated withvancomycin. There is significant concern that if S. aureus also becomesresistant to vancomycin, the health care profession will be left withouttreatment for these types of infections. Increasing resistance amongseveral types of Gram-positive bacteria associated with common andpotentially life-threatening infections complicate the treatment ofserious infections and has been linked to extended hospitalizations,higher medical costs and high mortality rates.

Like the family of β-lactam antibiotics, vancomycin acts onpeptidoglycan metabolism. The peptidoglycan is essential for bacterialsurvival because of its function as the exoskeleton that prevents cellrupture due to internal pressure. By binding to the D—Ala—D—Ala moietyof the bacterial cell wall precursors, vancomycin interferes with thegrowth of the peptidoglycan (6). In the resistant strains with vanA orvanB phenotype however, some of the D—Ala—D—Ala moiety of the cell wallprecursors is substituted by analogous D—Ala—D—Lac ones (7-9). Only asmall percentage of the enterococcus peptidoglycan layer is needed to bestructurally altered from D—Ala—D—Ala to D—Ala—D—Lac to cause anincrease in the vancomycin MIC (10% of the altered peptidoglycanincreases the MIC of vancomycin from 2 to 32 ug/ml).

Resistant bacteria carry a transportable element encoding nine genesthat contribute to the resistance phenotype (10). These gene productsinclude VanS, a transmembrane protein that senses directly or indirectlythe presence of vancomycin. Once autophosphorylated, VanS transmits asignal to a response regulatory protein VanR that activatestranscription of the other resistance genes (11). VanA is involved inthe synthesis of the depsipeptide D—Ala—D—Lac while VanH convertspyruvate into D-lactate. This pathway is essential for the resistancephenotype (12,13). VanX is a Zn²⁺ dependent pepsidase that selectivelycleaves D—Ala—D—Ala leading to an accumulation of the depsipeptide, andthus, of precursors with altered D—Ala—D—Lac termini (14,15). VanY is amembrane bound D—D-carboxypeptidase that hydrolyses the normal cell wallprecursor lipid-intermediates, further increasing the pool of precursorswith altered termini (16). However, the formation of D—Ala—D—Alacontinues in the cell due to the activity of the native enterococcalD—Ala—D—Ala ligase. Because vancomycin binds to D—Ala—D—Ala substrates,a mechanism is required to prevent D—Ala—D—Ala from being incorporatedinto the cell wall. VanX and vanY perform this function. As a result ofthe incorporation of D—Ala—D—Lac by vancomycin resistant enterococcus(VRE), the affinity of vancomycin for the peptidoglycan layer diminishesover 1000-fold, leading to antibiotic resistance. VanA strain is themost common phenotype of VRE and is described by inducible, high-levelresistance that is associated with the van genes that lead toD—Ala—D—Lac altered termini.

In order to bypass resistance, vancomycin has been modified to enhanceits binding to D—Ala—D—Lac and inhibitors of the D—Ala—D—Lacbiosynthetic pathway have been sought (17-19). Here we propose anotherapproach—the selective and catalytic cleavage of the D—Ala—D—Lacdepsipeptide by small molecules. By reducing the concentration ofprecursors with altered termini one would expect to re-sensitize thebacteria to vancomycin. A small molecule that performs such task couldbe used in concert with vancomycin (or vancomycin derivatives withhigher affinity) in the treatment of vanA resistant strains.

SUMMARY OF THE INVENTION

One of the most challenging situations for the immuno-compromisedpatients is the development of vancomycin-resistant enterococci (VRE),an increasingly frequent cause of hospital-acquired infections in theUnited States. These organisms are resistant to virtually all currentlyavailable antibiotics including vancomycin, considered the agent of lastresort for Gram-positive infections. VanA strain is the most commonphenotype of VRE and is described by inducible, high-level resistancethat is associated with the van genes that lead to D—Ala—D—Lac cell wallaltered termini. Here we describe the development of small moleculesthat catalytically and selectively cleave the altered termini of thebacteria cell wall so as to disable the antibiotic-resistance mechanismin these pathogens. The molecules re-sensitize bacteria to the drug andcould be used in concert with vancomycin in the treatment of VRE.

The invention provides a method of treating a subject afflicted with aninfection caused by glycopeptide antibiotic resistant Gram-positivebacteria, such as vancomycin resistant Gram-positive bacteria, in whichresistance results from the conversion of an amide bond to an ester bondin the cell wall peptide precursors of the bacteria which comprisesadministering to the subject an antibacterial amount of glycopeptideantibiotic, such as vancomycin or a homolog of vancomycin, and an amountof an agent effective to selectively cleave the ester bond so as tothereby treat the subject.

The invention also provides a method of killing glycopeptide antibioticresistant Gram-positive bacteria, such as vancomycin resistant Van A,Van B, Van D, or Van G Gram-positive bacteria which comprises contactingthe bacteria with an agent that selectively cleaves D—Ala—D—Lac cellwall depsipeptides in the bacteria in an amount effective to cleave suchdepsipeptides and an antibacterial amount of glycopeptide antibiotic,such as vancomycin or a homolog of vancomycin, so as to thereby kill thebacteria.

The invention further provides a method for determining whether a testcompound selectively cleaves an ester bond present between two aminoacid-like moieties in a depsipeptide which comprises contacting acompound comprising the structure X-Y, wherein each of X and Y are aminoacid-like moieties and — is an ester bond with the test compound anddetermining whether the test compound cleaves the ester bond.

In another aspect, the invention provides a method of treating a subjectafflicted with an infection caused by glycopeptide antibiotic resistantGram-positive bacteria in which resistance results from the conversionof an amide bond to an ester bond in the cell wall peptide precursors ofthe bacteria which comprises administering to the subject anantibacterial amount of a glycopeptide antibiotic and an amount of anagent effective to selectively cleave said ester bond so as to therebytreat the subject.

In addition, the present invention also provides a method of killingglycopeptide antibiotic resistant Gram-positive bacteria which comprisescontacting the bacteria with an agent that selectively cleavesD—Ala—D—Lac cell wall depsipeptides in the bacteria in an amounteffective to cleave such depsipeptides and an antibacterial amount ofthe glycopeptide antibiotic so as to thereby kill the bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Combinatorial libraries selected to screen for cleavage ofD—Ala—D—Lac.

FIG. 2. Computer generated model of the complex of BnNHL-Lys-D-Pro-L-Serdimethylurea 4 a with PhNH—D—Ala—D—Lac. Calculated hydrogen bonds aredepicted by dashed lines.

FIG. 3. Schematic representation of the small molecules assayed with thedepsipeptide and the intermediate resulted by the nucleophilic attack ofthe serine.

FIG. 4. Representative kinetic data for the hydrolysis of depsipeptidederivative 5 by 20 mM phosphate buffer (pH=7.0) (triangles),BnNHL-Lys-D-Pro-L-Ser dimethylurea 4 a (filled circle), control sequence6 (diamond), control sequence 7 (filled triangle), control sequence 8(box), control sequences 6+8 (circle). Assays were performed withdepsipeptide derivative 5 at 0.5 mM, while peptides were run at 12 mM.The graph is the average of five separate runs.

FIG. 5. Schematic representation of the S-prolinol derivatives.

FIG. 6. Representative kinetic data for the hydrolysis of depsipeptidederivative 5 by 20 mM phosphate buffer pH=7.0 (circles), SProDAla(squares), SProUC4 (diamonds), SProC1 (asterisks), SProC2 (crosses),SProC3 (triangles), SProC4 (filled circles), SProC5 (filled squares),SProC6 (filled diamonds).

FIG. 7. Structure of the complex of PhD—Ala—D—Lac with SProC5 ascalculated by molecular modeling. Hydrogen bonds are depicted by dashedlines.

FIGS. 8A-8D. Illustration of the specific synergistic effect of SProC5against the vanA strain EF228. A) Strain EF228 was grown in the presenceof increasing concentration of vancomycin with (open squares) or without(solid squares) the small molecule SProC5 (50 mM). Bactericidal activitywas estimated by determining the number of colony forming units per ml(CFU/ml) that survived the combined treatment of vancomycin and SProC5(solid bars). B) Two structurally related molecules SProC2 (solidsymbols) and SProC5 (open symbols) were compared for their relativesynergy with vancomycin. Their activities were concentration dependent(0, 5 10 and 50 mM, represented by square) with SProC5 being the mostefficient molecule mirroring the kinetic assays (see FIG. 6). C) SProC5specificity together with vancomycin against strain EF228 was tested bycomparing to its enantiomer RProC5 or its 5 carbon unit C5. Resultsrepresent the average of four independent experiments at the fixedconcentration of 100 mM. D) Strain JH2-2 was used as a representative ofvancomycin susceptible strains that do not synthesize alteredD—Ala—D—Lac terminating cell wall precursors.

FIGS. 9A-9B. (A) Picture of a combinatorial library after screeningagainst substrate 1 and removal of the solution (B) Picture of the samelibrary after several DMF washes.

FIG. 10. Photograph of library SSY before picking the red-label carryingactive beads. Assay carried out at 0.85 mM (1) in DMF. One red beadselected for analysis is pointed out in the upper-left corner of theimage.

FIG. 11. Methodology utilized for the synthesis of dye-labeleddepsipeptide (1)(a). 2-(Trimethylsilyl)ethanol, Ti(i-PrO)₄, THF; (b).BocD—AlaCOF, DIEA, DMAP, DCM; (c). TFA:DCM=4:1; (d). A, DIEA, DMAP, DCM;(e). TBAF; (f). Methyl 4-hydroxybenzoate, DEAD, PPh₃, toluene:DCM=5:1;(g). LiOH; (h). Cyanuric fluoride, pyridine, DCM.

FIG. 12. General procedure for the synthesis of compounds ProCn.

FIG. 13. Synthesis of SProUC4. (a) BocON, TEA; (b) phosgene, B,pyridine; (c) NaBH₄; (d) TFA.

FIG. 14. Representative kinetic data for the hydrolysis of depsipeptidederivative 5 by 20 mM phosphate buffer (pH 7.0) (triangles),BnNHL—Lys—D—Pro—L—Ser dimethylurea 4 a (filled circle), control sequence6 (diamond), control sequence 7 (filled triangle), control sequence 8(box), control sequences 6+8 (circle). Assays were performed withdepsipeptide derivative 5 at 0.5 mM, while peptides were run at 12 mM.The graph is the average of five separate runs.

FIG. 15. MS (FAB) of the THF-water assay mixture; m/z=491 (M+1) forBnNHL—Lys—D—Pro—L—Ser dimethylurea 4 a, 506 for D—Ala derivative 4, 578for substrate 1 and 978 for transesterification product 3.

FIG. 16. Schematic representation of the intermediate resulted by thenucleophilic attack of the BnNHL—Lys—D—Pro—L—Ser dimethylurea 4 a serineon analog 1 and the ¹H NMR model compound.

FIG. 17. Representation of the H¹ NMR spectra (A) of compound 9, (B) ofBnNHL—Lys—D—Pro—L—Ser 4 a and (C) of THF-5% water assay mixture.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of treating a subject afflicted with aninfection caused by glycopeptide antibiotic resistant Gram-positivebacteria, such as vancomycin resistant Gram-positive bacteria, in whichresistance results from the conversion of an amide bond to an ester bondin the cell wall peptide precursors of the bacteria which comprisesadministering to the subject an antibacterial amount of vancomycin or ahomolog of vancomycin and an amount of an agent effective to selectivelycleave the ester bond so as to thereby treat the subject.

In one embodiment of the invention, the subject is a human being.

In general, the agent is an activated nucleophile and is furthercharacterized by the presence within the agent of an electrophile andchirality complementary to a bacterial cell wall depsipeptide.

In one embodiment of the invention, the agent is represented by theformula S—Pro—C_(n).

In another embodiment of the invention, the agent has the structure:

wherein n is an integer from 1 to 6 inclusive and R is hydrogen or a C₁to C₆ straight chain or branched alkyl group.

Preferably, the agent catalytically cleaves the ester bond in the cellwall peptide precursors, for example, the ester bond in the structureD—Ala—D—Lac.

The agent may be administered prior to administering a glycopeptideantibiotic, such as vancomycin or the homolog of vancomycin.Specifically, the agent is administered a sufficient period of timeprior to administering vancomycin or the homolog of vancomycin to permitcleavage of the ester bond in the cell wall peptide precursors to beeffected.

Alternatively, the agent and glycopeptide antibiotic, such as vancomycinor the homolog of vancomycin are administered simultaneously, forexample, the agent may be covalently attached to vancomycin or thehomolog of vancomycin.

In the practice of the invention, the bacteria are typically Van A, VanB, Van D or Van G Gram positive bacteria.

In another preferred embodiment of the invention, the bacteria may beStaphylococcus bacteria, S. aureus bacteria, Enterococcus bacteria,Streptococcus bacteria, Leuconostoc bacteria, Pediococcus bacteria,Lactobacillus bacteria, and Erysipelothrix bacteria.

This invention also provides a method of killing vancomycin resistantVan A, Van B, Van D, or Van G Gram-positive bacteria which comprisescontacting the bacteria with an agent that selectively cleavesD—Ala—D—Lac cell wall depsipeptides in the bacteria in an amounteffective to cleave such depsipeptides and an antibacterial amount ofvancomycin or a homolog of vancomycin so as to thereby kill thebacteria.

In one embodiment of the invention, the invention provides a method ofkilling glycopeptide antibiotic resistant Gram-positive bacteria, suchas vancomycin resistant Van A, Van B, Van D, or Van G Gram-positivebacteria which comprises contacting the bacteria with an agent thatselectively cleaves D—Ala—D—Lac cell wall depsipeptides in the bacteriain an amount effective to cleave such depsipeptides and an antibacterialamount of glycopeptide antibiotic, such as vancomycin or a homolog ofvancomycin, so as to thereby kill the bacteria wherein the agent is anactivated nucleophile, and the agent is further characterized by thepresence within the agent of an electrophile and chirality complementaryto the bacterial cell wall depsipeptide.

In another embodiment of the invention, the agent maybe represented bythe formula S—Pro—Cn.

In one embodiment of the invention, the invention provides a method ofkilling glycopeptide antibiotic resistant Gram-positive bacteria, suchas vancomycin resistant Van A, Van B, Van D, or Van G Gram-positivebacteria which comprises contacting the bacteria with an agent thatselectively cleaves D—Ala—D—Lac cell wall depsipeptides in the bacteriain an amount effective to cleave such depsipeptides and an antibacterialamount of glycopeptide antibiotic, such as vancomycin or a homolog ofvancomycin, so as to thereby kill the bacteria, wherein the agent hasthe structure:

wherein n is an integer from 1 to 6 inclusive and R is hydrogen or a C₁to C₆ straight chain or branched alkyl group.

The invention provides a method of killing glycopeptide antibioticresistant Gram-positive bacteria, such as vancomycin resistant Van A,Van B, Van D, or Van G Gram-positive bacteria which comprises contactingthe bacteria with an agent that selectively cleaves D—Ala—D—Lac cellwall depsipeptides in the bacteria in an amount effective to cleave suchdepsipeptides and an antibacterial amount of glycopeptide antibiotic,such as vancomycin or a homolog of vancomycin, so as to thereby kill thebacteria, where the agent preferably, catalytically cleaves the esterbond in the D—Ala—D—Lac depsipeptide.

In the practice of the invention, the agent is administered prior toadministering the glycopeptide antibiotic, such as vancomycin or thehomolog of vancomycin, desirably a sufficient period of time prior toadministering vancomycin or the homolog of vancomycin to permit cleavageof the ester bond to be effected in the D—Ala—D—Lac depsipeptide.

Alternatively, the agent and the glycopeptide antibiotic, such asvancomycin or the homolog of vancomycin may be administeredsimultaneously, e.g. the agent may be covalently attached to vancomycinor the homolog of vancomycin.

In yet another embodiment, this invention provides a method fordetermining whether a test compound selectively cleaves an ester bondpresent between an amino acid and an α-hydroxy carboxylic acid in adepsipeptide which comprises contacting a compound of the structure X-Y,where X is an amino acid and Y is α-hydroxy carboxylic acid and — is anester bond, with the test compound and determining whether the testcompound cleaves the ester bond.

In yet another embodiment, the invention provides a method fordetermining whether a test compound selectively cleaves an ester bondpresent between two amino acid-like moieties in a depsipeptide whichcomprises contacting a compound comprising the structure X-Y, whereineach of X and Y are amino acid-like moieties and — is an ester bond withthe test compound and determining whether the test compound cleaves theester bond, for example where the ester bond is present in the structureD—Ala—D—Lac.

In an embodiment of invention, the compound comprises the structureL-(X-Y) wherein (X-Y) is D—Ala—D—Lac, and wherein L is a detectablelabel, for example a dye.

In one embodiment of the invention, the test compound is bound to asolid support.

In yet another embodiment of the invention, the test compound is presentin a collection of compounds containing nucleophiles, for example, acombinatorial library of compounds.

As used herein “homolog of vancomycin” refers to vancomycin having atleast one more CH₂ or alkene group in its molecule than the vancomycinmolecule. See for example U.S. Pat. No. 6,037,447, the contents of whichare hereby incorporated by reference into this application.

As used herein “glycopeptide antibiotic” refers to a class of compoundsdisclosed in, by way of example and not as a limitation to the presentinvention, U.S. Pat. No. 5,977,062, the contents of which are herebyincorporated by reference into this application. For example,glycopeptide antibiotics could include vancomycin as disclosed in U.S.Pat. No. 3,067,099; A82846A, A82846B, and A82846C as disclosed in U.S.Pat. No. 5,312,738; PA-42867 factors A, C, and D as disclosed in U.S.Pat. No. 4,946,941; A83850 as disclosed in U.S. Pat. No. 5,187,082;avoparcin as disclosed in U.S. Pat. No. 4,322,343; actinoidin, alsoknown as K288 (J. Antibiotics Series A 14:141 (1961)); helevecardin(Chem. Abstracts 110:17188 (1989); galacardin (Chem. Abstracts 110:17188(1989); and M47767 (PCT International Application No. WO 91/06566).

Organisms intrinsically resistant to vancomycin usually produceD—Ala—D—Lac. Theoretically, SProC5 alone could be bactericidal againstsuch bacteria(e.g., Leuconostoc, Pediococcus, Lactobacillus andErysipelothrix sp.) (34).

The following description and examples are presented to furtherillustrate and explain the present invention and should not be taken aslimiting in any regard. Unless otherwise indicated in the examples andelsewhere in the specification and claims, all parts and percentages areby weight. Temperatures are in degrees Centigrade.

EXPERIMENTAL DETAILS

Combinatorial Library Screening

To find small molecules that cleave the D—Ala—D—Lac depsipeptides, thered dye-labeled analog 1 is prepared as a probe (Scheme 1).

Scheme 1. a. 2-(Trimethylsilyl)ethanol, Ti(i-PrO)₄, THF; b. BocD—AlaCOF,DIEA, DMAP, DCM; c. TFA:DCM=4:1; d. A, DIEA, DMAP, DCM; e. TBAF; f.Methyl 4-hydroxybenzoate, DEAD, PPh₃, toluene:DCM=5:1; g. LiOH; h.Cyanuric Fluoride, pyridine, DCM.

Substrate 1 is treated with combinatorial libraries of potentialnucleophiles on solid phase synthesis beads (2), and then those librarymembers are selected that covalently linked the dye to a bead (3). Thiscould be possible assuming that noncovalently bound material would bewashed away with a polar solvent (Scheme 2).

Scheme 2. Strategy for active sequence determination.

The libraries used for screening (i.e., the libraries used in assayswere described in: (a) M. Burger, W. C. Still, J. Org. Chem. 60, 7382(1995); (b) A. Borchardt, W. C. Still, J. Am. Chem. Soc. 116, 373(1994). (c) Y. Cheng, T. Suenaga, W. C. Still, J. Am. Chem. Soc. 118,1813 (1996). (d) H. Wenemers, thesis, Columbia University (1996); (e) G.Li, thesis, Columbia University (1993); (f) E. J. Iorio, thesis,Columbia University (1999)), are presented in FIG. 1 and were randomlyselected from the Still group archive to achieve higher structuraldiversity. All libraries posses amino acid building blocks, however, thevariance rises from the scaffold that allows a structurally differentorganization of these building blocks. All peptides were side chaindeprotected and washed with DCM/TEA after TFA deprotection to ensurethat all possible nucleophiles were in the unprotonated state.

The assay for screening these libraries against 1 involves shaking adesired amount of library beads with the solution of labeled substratefor a period of time. To the library beads were added 200 μL solution ofsubstrate 1 in 12DCE (or DMF), and the mixture was rotated for 3-5 daysin a small glass vial. DMF was added and shaking continued for 1 h.Solvent was removed and the washing was repeated several times. One washwas performed with one drop of benzylamine added to DMF to remove anyremaining physically bound substrate. The strongly red beads were pickedindividually in 1.5 μL DMF (in 25 μL capillaries) and photolysed for 6 hunder a short wave UV lamp. Decoding was achieved by injecting thecontent of each capillary in EC-GC and comparing the chromatogram to astandard (20,21).

After the reaction occurred, the label-carrying beads were selected andanalyzed (20,21). The initial assays were performed in1,2-dichloroethane (12DCE) at a concentration of 2.3 mM in substrate 1.Results revealed that in every library the active sequences carriedserine at the amino-terminal position. This finding is remarkableconsidering that other nucleophiles such as Thr, Lys and terminal aminofunctionality, present in the screened libraries, did not appear at thatposition in the red beads.

Additionally:

for library GLPro, 80% of the active beads carried the sequenceD-Pro—L—Pro—L—Ser on the first arm (C3), while the other 20%Gly—L—Pro—L—Ser on the second arm (C7)

library MB3 showed activity only after previous equilibration withCu(OAc)₂. Position A₂A₃ was always occupied by the sequence Pro—Ser,while A₁ was somehow variable

library MB4 revealed only one active sequence:D—Asn—L—Lys—L—Pro—L—SerNH₂

library SSY carried exclusively the dimethylurea capping group from achoice of 14 others, and the most colored beads had frequently Pro andLys in a neighboring position to the terminal Ser

library Yuan Cheng exhibited activity only if initially equilibratedwith Cu(OAc)₂. Under those conditions, 85% of the active beads had thecalibration mark: trans L-hydroxyPro1-A₁-A₂cis,trans-L-hydroxyPro2-D-Ser-A₄

libraries HW and JW, acetylated tripeptide libraries, although carryinga larger selection of amino acids than SSY, showed no activity under theassay conditions

Control assays were performed with side chain protected libraries, andadditionally, with the trimethylsilyl ethyl ester of the substrate 1.Neither assay resulted in active sequences.

To improve selectivity in the case of library SSY, assays were performedin different solvents and lower concentrations. From a choice of 12DCE,DCM, THF and DMF, best results were obtained in DMF where selectivityand intensity of the beads were enhanced. A decrease in concentration ofthe substrate to 0.85 mM also improved selectivity. This concentrationproved to be the lower threshold for eye detection of red beads.

Under these conditions, three sequences were most prominently found:

X—L—Lys—L—Ser dimethylurea

X—D—Lys—D—Ser dimethylurea where X was variable and

L—Lys—D—Pro—L—Ser dimethylurea

After a careful analysis of the results, one can speculate that allactive sequences carry a nucleophile (Ser) and an electrophile (Lys inmost cases, Cu²⁺ for libraries Yuan Chen and MB3, probably a backbone NHfor library GLPro). These must be effectively oriented to make thenucleophilic attack of serine possible. The prevalence of Pro suggeststhat this amino acid may be involved in inducing conformational rigidityand therefore, pre-organization of the active sites.

Computer Modeling of the Active Peptides

To gain a better understanding of why these sequences were favored andhow they work, molecular modeling studies were carried out on selectedsequences and on the complexes of these sequences with D—Ala—D—Lac (FIG.2). Simulations were performed using the GB/SA solvation method forwater and AMBER* force field, as implemented in Macromodel V6.0. We usedMCMM (Monte Carlo Multiple Minimum) alternated with LMCS (Low FrequencyMode Conformational Search) as conformational search methods. We foundthat MCMM performed better for finding minima different from the initialconformations, while LMCS for minima close to the initial conformations.Generally, a search was started with MCMM, the output conformations werere-minimized and the conformations lower than 3 or 5 kcal were used asinput for a new search using LMCS until convergence was obtained. Thismethod was applied for the sequence BnNHL—Lys—D—Pro—L—Ser dimethylurea(4 a), an active sequence from library SSY.

Modeling supports the observations deducted from the combinatorialassays. The structural skeleton permits Lys and Ser to be in closeproximity. Lys is involved in binding the carboxylate of D—Ala—D—Lac inaddition to its role as the electrophile that stabilizes the tetrahedraltransition state. Additionally, the nucleophilicity of Ser is enhancedby hydrogen bonding to the urea capping group. This explains why thedimethyl urea capping group (the best hydrogen acceptor) was the onlyone that occurred in the active sequences resulted from the library SSY.Furthermore, the hydroxyl is favorably positioned for attacking theester group of the depsipeptide.

Study of the Electivity and Efficiency of these Peptides in CleavingD—Ala—D—Lac

To test the efficiency of these simple peptides in cleaving D—Ala—D—Lacunder physiologically relevant conditions, we chose studyingL—Lys—D—Pro—L—Ser dimethylurea, an active sequence found more frequentlyin the assays.

The peptide 4 a and the depsipeptide derivative 5 were prepared insolution (FIG. 3 and Scheme 2). The need to replace the substrate 1 with5 emerged because of the poor solubility of 1 in water.

The ability of peptide 4 a in cleaving the substrate 5 was assessed inaqueous phosphate buffer pH=7 at 37° C. Stock solutions of 16 mM of 5and 130 mM of peptide were made in water. Phosphate buffer solution of25 mM was made by adjusting the pH of a K₂HPO₄ solution to 7.0 byaddition of concentrated HCl. To a 1.5 mL glass vial (Waters) were added5 μL stock solution of 5 and 15 μL stock solution of peptide, followedby 135 μL buffer. The volume was adjusted to 160 μL by addition ofwater. Vials were kept in an incubator at 37° C. and 2 μL aliquots weretaken every 3 h. Each aliquot was diluted with water to 5 μL and 2 μLwere injected in HPLC. Separation of components was carried out on areverse phase column C18 (Waters) using a gradient water/acetonitrile(0.1% TFA).

Using HPLC and monitoring the p-NO₂-phenyl derivative by UV at 275 nm,we could easily follow the disappearance of D—Ala—D—Lac derivative 5 andthe formation of the hydrolyzed product 4. Under these conditions, weobserved a 20% cleavage of the depsipeptide in 24 hrs. No significanteffect on the rate of hydrolysis over buffer was observed using thecontrol sequences 6, 7, 8 alone or 6 and 8 combined (FIG. 4). Thisproves that the whole structural assembly is necessary for the reactionto occur and that the reaction is not an artifact resulted by a changein the pH of the media due to the presence of the amine functionality.

The enantiomer of 4 a was synthesized through the same procedure and itsability to cleave the depsipeptide was measured. The data obtained (notshown) suggests that the enantiomer is less than half as active as 4 a.This observation explains why the enantiomeric peptide D—Lys—L—Pro—D—Serdimethylurea was never found as an active sequence in the combinatoriallibrary assays. The presence of a well-oriented assembly of anucleophile and electrophile is essential but not sufficient for thereaction to occur; chiral complementary between the depsipeptide and thecleaving molecule is also required and suggests the formation of acomplex between the two molecules prior to the cleavage of the ester.

Mechanistic Studies

To prove that the reaction occurs via a nucleophilic attack by serine,the cleavage of 1 by 4 a was studied in THF-5% water, media in which thetransesterification product 3 could be observed (Scheme 3).

Scheme 3. Proposed mechanism for the catalytic cleavage of D—Ala—D—Lacby 4 a.

The reaction could be monitored by HPLC at 485 nm, and the separation ofthe three components was easily performed on an analytical reverse phasecolumn using a gradient of acetonitrile:water. Stock solutions of 2 mMconcentration of 1 in THF and 49 mM of 4 a (lyophilized from PIPESbuffer pH=7.0) in water were prepared. In three ampoules were added 40μL solution of 1, 7 μL of 4 a and the volume was adjusted to 160 μL withTHF. For background measurements, in another three ampoules 7 μL waterwere added instead of 4 a to the solution of 1. All six ampoules weresealed under an argon stream and placed in an oil bath heated at 60° C.For each measurement one vial was opened and 5 μL were taken, dilutedwith 5 μL THF and 2 μL of this solution were injected in the HPLC.Isolation of the intermediate 3 proved to be however, more difficult.Application of the assay mixture to a size exclusion column (SephadexLH-20 with DMF) gave a fraction enriched in 3, and this was used for aCOSY-¹H NMR analysis (FIG. 16). Comparison of the NMR spectra of 4 a, 3and 9 confirmed the identity of the intermediate 3 (FIG. 17). Massspectrum analysis (MS) was used to additionally establish the identityof the three peaks seen in the HPLC chromatogram (FIG. 15).

Small Molecule Development

The peptides resulted from the non-biased combinatorial librariesscreenings are not useful as therapeutic agents due to their lowcatalytic activity. Additionally, they are easily destroyed byproteases. The goal of such screens was to gain an understanding of thekey elements required for selective and catalytic cleavage of thealtered termini and then assemble them in a simple structure.

If our observations are correct, a small molecule that has a hydroxyl(serine-like functionality) of enhanced nucleophilicity, a well-orientedelectrophile and a complementary chirality to the depsipeptide shouldcatalyze the cleavage of the D—Ala—D—Lac with an efficacy comparable tothe small peptide 4 a. N-acylated prolinol derivatives (FIG. 5) are thesimplest structures that could fulfill such requirements. Their primaryalcohol functionality forms an internal H-bond with the amide, thestructures allow for the addition of the electrophile (NH₂) throughvarious linkers and moreover, are chiral molecules. The derivatives weretested for their ability to cleave 5 in aqueous phosphate buffer pH=7.0at 37° C. (FIG. 6). Addition of 12 mM SProC5 induces the cleavage of 50%of depsipeptide in 24 hrs, implying that the SProC5 derivative is twiceas active as the initial peptide 4 a. One explanation for the higheractivity of the very simple molecule SProC5 compared to 4 a is theenhanced nucleophilicity of its hydroxyl (H position in H¹ NMR 7.59 ppmvs 6.62 ppm). Activity declines in the series with the decrease of thechain length from 5 carbons to 1 carbon. This result can be explainednot only by a decrease in efficiency of the terminal amino in reachingto the carboxylate and ester of D—Ala—D—Lac with the shortening of thechain, but also by the decrease in nucleophilicity of the hydroxyl. A 6carbon chain is also less active. The lower activity of SProC6 isprobably due to the higher flexibility of the carbon chain that does notrender the amino group available for the reaction. To confirm thesespeculations we compared the chemical shifts of the OH and NH in the H¹NMR spectra of the NHBoc protected SProCn series (Table 1).

Substrate 5 was used at 0.5 mM, while prolinol derivatives were run at12 mM. The graph is the average of three separate assays (FIG. 6).

The study shows that there is a competition between the amino group andthe hydroxyl for hydrogen bonding to the amide. With the decrease of thechain length, the probability of the OH being hydrogen bonded decreasessubstantially, fact reflected in the chemical shifts of the OH and NHwith the change in the length of the carbon chain.

TABLE 1 Shifts in the position of OH and NHBoc with the modification ofthe carbon chain length Boc- NH position derivative OH position (ppm)(ppm) SProC1 4.57 5.48 SProC2 4.98 5.28 SProC3 4.96 4.75 SProC4 5.114.65 SProC5 5.14 4.54 SProC6 5.28 4.52 SProUC4 4.86 4.65

The table also explains the low activity of SProUC4, derivative in whichthe amide is replaced by urea. The urea is a better acceptor than theamide and should increase the reactivity of the hydroxyl. However, it ispossible that structural constraints imposed by the urea play animportant role and do not allow for proper orientation for H-bonding.

Molecular modeling performed on the complex of SProC5 and PhD—Ala—D—Lacadditionally confirms the structural fit of this small molecule for thecleavage of the depsipeptide (FIG. 7).

In vivo Testing of the Small Molecules against VRE

The designed small molecules should theoretically enhance the biologicalactivity of vancomycin by reducing the pool of C-terminal altered cellwall precursors in bacteria that have intrinsic or acquired resistanceto vancomycin. Therefore, to test the activity of the syntheticmolecules, we used as reference organism a vanA enterococci, strainEF228 (22), and for comparison, the susceptible reference strainenterococci JH2-2 (23). SProC5, the best candidate molecule was studiedin combination with vancomycin. Enterococcus faecium EF228 (22), a vanAstrain and Enterococcus faecalis strains JH2-2 (23) were grown on BHI(Difco or Oxoid) agar or broth at 37° C. Biological assays wereperformed in 96-well tissue culture plates (MICROTEST U-bottom, Falcon,Becton Dickinson). A range of vancomycin (Sigma) concentrations (100 μlper well) were used based on sequential two-fold dilution starting from100 and 2000 μg/ml to 0.1 and 1.96 μg/ml for strains JH2-2 and EF228,respectively. The different molecules were added (100 μl per well) atfixed concentrations (0, 5, 10, 50 and 100 mM) containing an inoculum ofeither strain at a final dilution of 10⁻² obtained from an overnightculture. The range of effective vancomycin concentrations started at 50and 1000 μg/ml for JH2-2 and EF228, respectively. Micro-titer plateswere incubated at 37° C. without agitation for 18 hours. Cell sedimentswere resuspended by shaking and optical density at 600 nm was measuredwith an ELISA Multiskan RC plate reader (Labsystems, Helsinki, Finland).Bactericidal activity was determined by serially diluting (10⁻², 10⁻⁴and 10⁻⁵) each well in BHI broth and plating 10 μl of each dilution onBHI agar plates. Plates were incubated 24 hours and the number of colonyforming units per ml was determined.

A reduction of 10% in the load of altered termini should theoreticallydecrease the MIC of vancomycin by 10-fold.

FIG. 8 illustrates the specific synergistic effect of SProC5 against thevanA strain EF228. Panel A shows that vancomycin alone inhibited growthat 500 μg/ml while SProC5 (50 mM) combined with vancomycin reduced theminimum inhibitory concentration to 62.5 μg/ml, i.e. a 8-16 foldsdecrease in the MIC. Bactericidal activity was confirmed by determiningthe number of cells that survived the combined treatment of vancomycinand SProC5 (FIG. 8A, black bars). Indeed, the combination of 62.5 μg/mlof vancomycin with 50 mM of SProC5 resulted in a three log decrease inbacterial load compared to vancomycin or SProC5 alone. This value wasincrease to four log when 250 μg/ml of vancomycin was used with 50 mMSProC5. The synergistic effect of SProC5 was dose dependent as shown inFIG. 8B (5 mM of SProC5 was ineffective while 10 mM had an intermediateactivity).

Specificity of SProC5 mode of action against D—Ala—D—Lac termini isreinforced by the complete absence of increased sensibility of JH2-2 tovancomycin by SProC5, SProC2 or any of the control molecules, even at100 mM. Results represent the average of two independent experiments(FIG. 8)

SProC5 alone had no inhibitory or bactericidal activity againstenterococci (see FIG. 8). Its synergistic effect with vancomycin couldbe related to a distinct mechanism than the one predicted based on thespecific hydrolytic activity of SProC5. To validate the mechanism, wecompared SProC5 activity to a related molecule SProC2 characterized by alower D—Ala—D—Lac hydrolytic activity in our kinetic assays (see FIG.6). Indeed, as predicted from the hydrolytic activity, SProC2 had a muchlower synergistic effect with vancomycin although not negligible (at 50mM, MIC to vancomycin decreased to 250 μg/ml). SProC2 was able todecrease the MIC to vancomycin only by 2 to 4 folds in the conditionstested (see FIG. 8B).

Further evidence correlates the hydrolytic activity of SProC5 with itssynergy with vancomycin. SProC5 activity was compared to that of itsenantiomer molecule RProC5 and to its corresponding 5 carbon unit (C5).None of the control molecules had a synergistic effect with vancomycineven at 100 mM (see FIG. 8C) strongly suggesting that the basis for thebiological activity of SProC5 was derived from its enhanced and specificD—Ala—D—Lac hydrolytic activity.

Finally, strain JH2-2 which is susceptible to vancomycin and does notsynthesize altered cell wall precursors, was used as a control. Thesensitivity of JH2-2 to vancomycin was unaffected by the presence of anyof the synthetic molecules (SProC2, C5, RProC5 and SProC5) even at 100mM (FIG. 8D). MIC of vancomycin was unchanged in any of the testedconditions (MIC between 1.58 and 3.13 μg/ml).

Through screening of combinatorial libraries and the study of the activesequences and patterns, an understanding of the essential key elementsfor catalytic and selective cleavage of D—Ala—D—Lac is achieved.Assimilation of these features resulted into the design of a simplesmall molecule that is more effective in cleaving the depsipeptide thanits paternal small peptide 4 a. This molecule, SProC5, increases thesensitivity of vanA resistant bacterial strain to vancomycin. Our invivo results suggest that SProC5 enhances vancomycin's activity becauseof its D—Ala—D—Lac's hydrolytic activity. The synergistic effect ofSProC5 mirrors its ability to hydrolyze the depsipeptide bond. Severalobservations support this hypothesis. First, a related compound SProC2,which has a lower hydrolytic activity, is less efficient in enhancingvancomycin's inhibitory activity as predicted from the kinetic studies.Furthermore, the enantiomer of SProC5—RProC5—lacks any synergy withvancomycin. Finally, the enterococcal strain JH2-2 which is unable tosynthesize altered cell wall precursors was completely insensitive tothe activity of the synthetic molecules. Taken together, these datasuggest that the mechanism of synergy in vivo is based on the hydrolyticcapability of the synthetic compound SProC5.

SProC5 can reverse resistance in several ways. It could similarly asVanX to reduce the cytoplasmic pool of D—Ala—D—Lac at every step of thebiosynthetic pathway, therefore favoring the synthesis of normal cellwall precursors terminating in D—Ala—D—Ala.

Alternatively, SProC5 could remain extracellular and actively hydrolyzecell wall lipid-intermediates. Lipid intermediates would be truncated asdisaccharide-tetrapeptides, an accumulation which would resultprogressively in a hypocrosslinked peptidoglycan. E.coli mutants thatare unable to properly recycle tetrapeptide turnover products,accumulate tetrapeptide derivatives resulting in lysis and death instationary phase (24).

A third mechanism can be envisioned. The resistance phenotype in vanAstrains is inducible and dependent on a two-component regulatory system.VanS and VanR function as a sensor and a response regulator,respectively. The suggested signal sensed by VanS appears be theaccumulation of lipid II (undecaprenyl-disaccharide-pentatpeptide)(25-27). The accumulation of tetrapeptide derivatives of lipid II due toSProC5 hydrolytic activity might compete for the binding site of VanSinterfering with the signaling cascade and the induction of vanHAXYZtranscription.

The three hypotheses for the mode of action of SProC5 are not exclusiveand could occur at the same time. A combination of muropeptide, cellwall precursor composition analysis and transcription analysis of thevanA cluster would be needed to distinguish between the differentmechanisms.

Molecules that catalytically and selectively cleave the altered terminiof the bacteria cell wall can disable the antibiotic-resistancemechanism in these pathogens. The molecules act by re-sensitizingbacteria to the drug and could be used in concert with vancomycin in thetreatment of VRE. Additionally, this work shows that bits of informationobtained from the screening of non-biased random libraries and frommolecular modeling can be assimilated in the design of structurallydifferent molecules that act by the same mechanism. One can envisionthat a more potent candidate for the cleavage of the D—Ala—D—Lac couldresult from screening biased libraries that assemble the structuralcharacteristics described in this work. We believe the approach foridentifying novel molecules able to enhance the activity of vancomycinhas long term potential for the management of infectious diseases.

Combinatorial Library Assay Development

The libraries used in screening for D—Ala—D—Lac cleavers are presentedin FIG. 1 and were randomly selected from the Still group archive toachieve higher structural diversity. All libraries posses amino acidbuilding blocks, however, the variance rises from the scaffold thatundoubtedly allows a structurally different organization of thesebuilding blocks. Library GLPro (28) uses 12-deoxycholic acid scaffoldthat allows differential derivatization of the two arms due to thedistinct reactivity of the C3 and C7 hydroxyls, while library Yuan Chen(29) employs, in addition to the cholic acid core, derivatizedhydroxyprolines to which the amino acids are linked. The cyclen corescaffold used for libraries MB3 and MB4 (30) contains three NH's asstarting points for growing the identical three or four amino acidpeptide chains, respectively. Libraries SSY (31), HW (32) and JW (33)are acylated tripeptide libraries linked to the solid support through acaproic acid unit. However, library SSY contains 15 different acylatinggroups, while all the members of libraries HW and JW are acetylated. Allpeptides were side chain deprotected and washed with dichloromethane(DCM)/triethylamine (TEA) after trifluoroacetic acid (TFA) deprotectionto ensure that all possible nucleophiles were in the unprotonated state.

The assay for screening these libraries against 1 involved shaking adesired amount of library beads with the solution of labeled substratefor a period of time. After the reaction occured, the label-carryingbeads were selected and analyzed. The initial assays were performed in1,2-dichloroethane (12DCE) at a concentration of 2.3 mM in substrate 1.After 3 days of shaking, the beads were extensively washed withdimethylformamide (DMF) and once with a diluted solution of benzylaminein DMF to remove any physically bound substrate (FIGS. 9A and 9B).

To improve selectivity in the case of library SSY, assays were performedin different solvents and lower concentrations. From a choice of DCE,DCM, tetrahydrofuran (THF) and DMF, best results were obtained in DMFwhere selectivity and intensity of the beads were enhanced (FIG. 11). Adecrease in concentration of the substrate to 0.85 mM also improvedselectivity. This concentration proved to be the lower threshold for eyedetection of red beads.

Results revealed that in every library the active sequences carriedserine at the amino-terminal position. This finding is remarkableconsidering that other nucleophiles such as Thr, Lys and terminal aminofunctionality, present in the screened libraries, did not appear at thatposition in the red beads.

Additionally:

for library GLPro, 80% of the active beads carried the sequenceD-Pro—L—Pro—L—Ser on the first arm (C3), while the other 20%Gly—L—Pro—L—Ser on the second arm (C7)

library MB3 showed activity only after previous equilibration withCu(OAc)₂. Position A₂A₃ was always occupied by the sequence Pro—Ser,while A₁ was somehow variable

library MB4 revealed only one active sequence:D—Asn—L—Lys—L—Pro—L—SerNH₂

library SSY carried exclusively the dimethylurea capping group from achoice of 14 others, and the most colored beads had frequently Pro andLys in a neighboring position to the terminal Ser

library Yuan Cheng exhibited activity only if initially equilibratedwith Cu(OAc)₂. Under those conditions, 85% of the active beads had thecalibration mark: trans L-hydroxyPro1A₁-A₂cis,trans-L-hydroxyPro2-D—Ser—A₄

libraries HW and JW, acetylated tripeptide libraries, although carryinga larger selection of amino acids than SSY, showed no activity under theassay conditions

Control assays were performed with side chain protected libraries, andadditionally, with the trimethylsilyl ethyl ester (TMSE) of thesubstrate 1. Neither assay resulted in active sequences.

Combinatorial Assay Sequence Data

TABLE 2 Library GLPro. 0.5 copies were used (about 10,000 beads) for a 3days assay in 12DCE with substrate 1. 15 red beads found. L, DPro A₁ A₂A₃ A₄ DPro Pro Ser Thr Lys DPro Pro Ser Lys Ala DPro Pro Ser Thr ProDPro Pro Ser Thr Ser DPro Pro Ser Lys Val DPro Pro Ser Pro Val DPro ProSer Val Ser DPro Pro Ser Thr Pro DPro Pro Ser Phe Ser DPro Pro Ser LysAla DPro Pro Ser Pro Ser DPro Pro Ser Pro Ser DPro Thr Pro Pro Ser DProPhe Ser Pro Ser LPro Leu Ala Pro Ser

TABLE 3 Library MB3. 0.1 copies were used, about 900 beadspre-equilibrated with Cu(OAc)₂, in an assay performed in 12DCE withsubstrate 1. 7 red beads found. A₁ A₂ A₃ LAla DPro DSer LVal DPro DSerLPro LPro LSer DAsn DPro LSer LVal DPro LSer LAla LPro LSer LGln DProDSer

TABLE 4 Library MB4. 0.07 copies were used (about 9,000 beads) used foran assay performed in DMF with substrate 1. 1 red bead found. A₁ A₂ A₃A₄ DAsn LLys LPro LSer

Tables 5A-5C. Library SSY. (A) 0.2 copies used, about 10,000 beads, foran assay in 12DCE with substrate 1. (B) 0.2 copies used, about 10,000beads, for an assay in DMF with substrate 1. (C) 1.5 copies used, about80,000 beads, for an assay in DMF with 0.85 mM substrate 1. Only thevery strongly red beads picked.

TABLE 5A A₁ A₂ A₃ CAP LLys DPro LSer NMe₂ LSer LLys LSer NMe₂ DPro LLysLSer NMe₂ DSer LLys LSer NMe₂ LPro LLys LSer NMe₂ LAla DLys DSer NMe₂DPro LPro DSer NMe₂ LLys LAla LSer NMe₂ DLys LVal DSer NMe₂ LLys DAlaLSer NMe₂ DLys LAla LSer NMe₂

TABLE 5B A₁ A₂ A₃ CAP LLys DPro LSer NMe₂ LAsn DPro LSer NMe₂ LLys LProDSer NMe₂ LPro LLys LSer NMe₂ DGln LLys LSer NMe₂ LSer LLys LSer NMe₂LLys DLys DSer NMe₂ LAla DLys Dser NMe₂

TABLE 5C Nr. of times A₁ A₂ A₃ CAP found LLys DPro LSer NMe₂ 2 DAsn LLysLSer NMe₂ 3 DPro LLys LSer NMe₂ 1 LAsn LLys LSer NMe₂ 1 DSer LLys LSerNMe₂ 2 LPro LLys LSer NMe₂ 1 DAla LLys LSer NMe₂ 1 LVal DLys DSer NMe₂ 1LAsn DLys DSer NMe₂ 1 DPro DLys DSer NMe₂ 1 DPro DAsn DSer NMe₂ 1 LProDGln DSer NMe₂ 1

TABLE 6 Library Yuan Chen. 0.2 copies used, about 8,000 beadspre-equilibrated with Cu(OAc)₂, for an assay done in 12DCE with 1.17 redbeads found Hp1 A₁ A₂ Hp2 A₃ A₄ trans L DPhe LPro cis L DSer DAla transL DPro LPhe cis L DSer LPro trans L DPro DSer trans L DSer LPhe trans LDPhe DAsn trans L DSer LPro trans L DAla LPro cis L DSer DPro trans LLAsn DPro cis L DSer LAla trans L LPro LPhe cis L DSer LAla trans L DProLPhe cis L DSer LPro trans L DSer LAla trans L DSer LPhe trans L DSerLAsn trans L DSer DPro trans L DAla LAla trans L DSer DSer trans L DAlaDPro trans L DSer DSer trans L LAla DPhe trans L DSer LAla cis L DAlaDSer trans L DSer DSer cis L LSer DSer trans D DAla DSer trans D LProDPro cis L DSer DSer

General Synthetic Procedures

All compounds were synthesized using standard laboratory techniques.Commercially available reagents and solvents were used without furtherpurification. Reactions were monitored using thin-layer liquidchromatography (TLC) and visualization was done using UV light, ceriumammonium molybdate (CAM) and permanganate.

Nuclear magnetic resonance (NMR) spectra were recorded on a VarianVXR-400, Bruker 400 MHz or Bruker 300 MHz. Mass spectra were obtainedwith Jeol JMS-HX110A Mass Spectrometer and RIBERMAG R10-10 C. Gaschromatography was conducted with a HP 5890 GC equipped with an electroncapture detector and HP ULTRA I fused silica capillary column. HPLC wasdirected with a Waters Millennium system, using a Nova Pak C18 column.

General Procedure for the Synthesis of Acid Fluorides:

To a 1 mmol solution of acid in 15 mL DCM at r.t. were added 1 eq.pyridine and 1.5 eq. cyanuric fluoride. The resulting solution wasstirred under argon for 1.5 h. After dilution with 150 mL DCM, theorganic layer was washed with 2 mL water. The solvent was removed togive the acid fluorides, which were used without further purification.

FIG. 11. Step (a) Synthesis of R(+)-2-(Trimethylsilyl)ethyl lactate. Asolution of 460 μL (4.8 mmols) of R-methyl lactate, 1.6 mL (5.38 mmols)Ti(i-PrO)₄ and 820 μL (5.7 mmols) 2-(trimethylsilyl)ethanol in 30 mL dryTHF was refluxed under argon overnight. Solvent was removed.Purification on column chromatography (DCM:acetone at 8:1) gave 700 mgof product in 85% yield.

¹H NMR (400 MHz, CDCl₃): δ4.28 (m, 2H), 4.22 (m, 1H), 2.82 (d, J=3.0 Hz,1H), 1.41 (d, J=7.3 Hz, 3H), 1.02 (m, 2H), 0.05 (s, 9H). Chemical ImpactMass Spectrum (CIMS) (NH₃): M=190 calculated for C₈H₁₈O₃. Found m/z=191(M+1).

FIG. 11. Steps (b, c). Synthesis of D—Ala—R(+)-2-(Trimethylsilyl)ethyllactate. To a solution of 700 mg (3.68 mmols) D-LacTMSE in 15 mL DCM,were added 800 mg (4.2 mmols) of the acid fluoride of D—AlaBoc, 1.2 mL(7.4 mmols) N,N-diisopropylethylamine (DIEA) and a catalytic amount ofdimethylaminopyridine (DMAP). The mixture was stirred at r.t. underargon for 3 h. Solvent was removed, and purification on columnchromatography (DCM:Petroleum ether:EtOAc at 10:4:1) gave 900 mg ofBocD—Ala—R(+)-2-(Trimethylsilyl)ethyl lactate (90% yield).

¹H NMR (400 MHz, CDCl₃): δ5.15-5.08 (m, 1H), 5.05-4.97 (bd, 1H),4.42-4.37 (m, 1H), 4.27-4.19 (m, 2H), 1.51 (d, J=7.1 Hz, 3H) 1.47 (d,J=7.3 Hz, 3H), 1.45 (s, 9H) 1.05-0.96 (m, 2H), 0.05 (s, 9H). ¹³C NMR(300 MHz, CDCl₃): δ173.3, 170.9, 155.5, 80.3, 69.6, 64.3, 49.4, 28.7,18.9, 17.7, 17.2, −1.1. CIMS (NH₃): M=361 calculated for C₁₆H₃₁O₆NSi.Found m/z=362 (M+1).

To this material were added 15 mL mixture DCM:TFA at 3:1, and thesolution was stirred at r.t. for 15 min. After solvent removal, the TFAsalt was used in the next step without further purification.

FIG. 11. Steps (f, g). A solution of 400 mg (1.26 mmols) Disperse Red 1,232 mg (1.52 mmols) Methyl-(4-hydroxy) benzoate, 434 mg (1.76 mmols)PPh₃ and 400 μL (2.52 mmols) diethyl azodicarboxylate (DEAD) in 25 mLtoluene/5 mL DCM, was stirred overnight under argon, at r.t. Solvent wasremoved. 200 mL DCM were added and the solution was washed with 3×15 mL10% NaOH. Purification on column chromatography (DCM) gave the methylester as a red solid.

¹H NMR (400 MHz, CDCl₃): δ8.34 (d, J=9.1 Hz, 2H), 7.99 (d, J=8.8 Hz,2H), 7.94 (d, J=9.0 Hz, 2H), 7.93 (d, J=9.1 Hz, 2H), 6.92 (d, J=8.8 Hz,2H), 6.83 (d, J=9.0 Hz, 2H), 4.26 (t, J=5.9 Hz, 2H), 3.90 (t, J=5.9 Hz,2H), 3.89 (s, 2H), 3.63 (q, J=7.1 Hz, 2H), 1.31 (t, J=7.1 Hz, 3H). CIMS(NH₃): M=448 calculated for C₂₄H₂₄O₅N₄. Found m/z=449 (M+1).

To this material were added 20 mL MeOH/40 mL THF/10 mL H₂O followed by 5equivalents of LiOH, and the solution was refluxed for 3 h. Solventremoved. After addition of 200 mL DCM, HCl concentrated solution wasslowly added until the solid has dissolved. The organic layer was washedwith 2×10 mL water. Solvent was removed to give 475 mg product in 83%overall yield. This was used without further purification.

¹H NMR (400 MHz, dimethylsulfoxide-d₆ (DMSO)): δ8.37 (d, J=9.1 Hz, 2H),7.94 (d, J=9.1 Hz, 2H), 7.87 (d, J=8.8 Hz, 2H), 7.85 (d, J=9.0 Hz, 2H),7.05 (d, J=8.8 Hz, 2H), 6.97 (d, J=9.0 Hz, 2H), 4.28 (t, J=5.9 Hz, 2H),3.92 (t, J=5.9 Hz, 2H), 3.63 (q, J=7.1 Hz, 2H), 1.20 (t, J=7.1 Hz, 3H).¹³C NMR (300 MHz, DMSO): δ168.1, 162.4, 157.1, 152.5, 147.7, 143.7,132.2, 126.9, 125.1, 123.4, 118.9, 114.9, 112.6, 66.6, 49.9, 46.2, 12.9.CIMS (NH₃): M=434 calculated for C₂₃H₂₂O₅N₄. Found m/z=435 (M+1).

FIG. 11. Step (h). To a solution of 265 mg (0.61 mmols) carboxylic acidin 10 mL DCM were added 53 μL (0.61 mmols) pyridine and 59 μL (0.8mmols) cyanuric fluoride, and the mixture was stirred under argon atr.t. for 1.5 h. The solution was diluted with 150 mL DCM and washed 1×2mL water. Solvent was removed to give the crude product.

FIG. 11. Step (d) Synthesis ofDR1-PhNH—D—Ala—D-Lactate-2-(ethyl)trimethylsilyl. The acid fluorideobtained as above, was taken in 20 mL DCM and added to the TFA salt ofthe amine, followed by 190 μL (1.1 mmols) DIEA and a catalytic amount ofDMAP, and the solution was stirred at r.t. under argon for 3 h. Solventwas removed and the product was purified by column chromatography(DCM:acetone at 15:1) to give 300 mg of red solid, in 80% yield.

¹H NMR (400 MHz, CDCl₃): δ8.33 (dd, J=8.0 Hz, J=1.9 Hz, 2H), 7.93 (dd,J=8.0 Hz, J=1.9 Hz, 2H), 7.92 (dd, J=8.0 Hz, J=2.1 Hz, 2H), 7.76 (dd,J=9.3 Hz, J=2.0 Hz, 2H), 6.92 (dd, J=8.0 Hz, J=2.1 Hz, 2H), 6.82 (dd,J=9.3 Hz, J=2.0 Hz, 2H), 6.59 (d, J=6.8 Hz, 1H), 5.20-5.11 (m, 1H),4.90-4.82 (m, 1H), 4.28 (m, 4H), 3.90 (t, J=5.9 Hz, 2H), 3.62 (q, J=6.9Hz, 2H), 1.61 (d, J=7.2 Hz, 3H), 1.54 (d, J=7.0 Hz, 3H), 1.29 (t, J=6.9Hz, 3H), 1.05-0.99 (m, 2H), 0.05 (s, 9H). Low resolution mass spectrum(LRMS) fast atom bombardment (FAB): M=677 calculated for C₃₄H₄₃O₈N₅Si.Found m/z=678 (M+1).

FIG. 11. Step (e). Synthesis of depsipeptide analogDR1-PhNH—D—Ala—D-Lactatic acid (1). To a solution of 300 mg (0.44 mmols)of trimethylsilylether (TMSE) protected 1 in 15 mL DMF, were added 440μL (0.44 mmols) solution 1 M tetrabutylammonium fluoride (TBAF) in THF.Solution was stirred for 1 h at r.t. After solvent removal the solid wastaken in 150 mL of EtOAc, acidified with acetic acid and washed with3×10 mL water. The solvent was removed, and purification by gelchromatography (Sephadex LH-20) with MeOH (1% AcOH) gave 254 mg 1 as ared solid in 80% yield over the last two steps.

¹H NMR (400 MHz, CDCl₃): δ8.33 (dd, J=8.0 Hz, J=1.9 Hz, 2H), 7.93 (dd,J=8.0 Hz, J=1.9 Hz, 2H), 7.92 (dd, J=8.0 Hz, J=2.1 Hz, 2H), 7.76 (dd,J=9.3 Hz, J=2.0 Hz, 2H), 6.92 (dd, J=8.0 Hz, J=2.1 Hz, 2H), 6.82 (dd,J=9.3 Hz, J=2.0 Hz, 2H), 6.62 (d, J=6.8 Hz, 1H), 5.24-5.15 (m, 1H),4.86-4.78 (m, 1H), 4.24 (t, J=5.9 Hz, 2H), 3.90 (t, J=5.9 Hz, 2H), 3.62(q, J=6.9 Hz, 2H), 1.60 (d, J=7.2 Hz, 3H), 1.59 (d, J=7.0 Hz, 3H), 1.29(t, J=6.9 Hz, 3H). ¹³C NMR (300 MHz, DMSO): 172.4, 171.6, 165.6, 160.7,156.1, 151.5, 146.7, 142.7, 129.3, 125.9, 124.8, 122.4, 113.8, 111.6,68.5, 65.5, 48.9, 47.9, 45.2, 16.6, 16.5, 11.9. High resolution massspectrum (HRMS) (FAB): calculated for C₂₉H₃₁O₈N₅ (M+1) 578.2251, found578.2255. Infrared analysis (IR) (polyethylene card): 3300, 3010, 2917,2950, 1746, 1735, 1710, 1604, 1513, 1339, 1253.

Synthesis of 4-Nitro-PhNHD—Ala—D—Lac (5). To the TFA salt solution ofD—Ala—D—LacTMSE in 10 mL DCM were added 2 equivalents of DIEA, acatalytic amount of DMAP and 1.3 equivalents of acid fluoride of the4-nitrobenzoic acid dissolved in 10 mL DCM. The solution was stirred for3 h at r.t. under argon. Solvent was removed and the product separatedon silica gel column with DCM:hexanes:acetone at 7:2:1. Deprotection ofthe TMSE was performed as previously described, and after purificationon Sephadex LH-20 (MeOH −1% AcOH), 5 was obtained as a white powder in80% overall yield.

¹H NMR (400 MHz, DMSO): δ9.17 (d, J=6.8 Hz, 1H), 8.38 (d, J=7.9 Hz, 2H),8.11 (d, J=7.9 Hz, 2H), 5.02-4.96 (m, 1H), 4.61-4.51 (m, 1H), 1.49 (d,J=7.3 Hz, 3H), 1.43 (d, J=7.0 Hz, 3H). ¹³C NMR (300 MHz, DMSO): δ172.8,172.2, 165.7, 150.0, 140.1, 129.8, 124.4, 69.7, 49.1, 17.6, 17.3. HRMS(FAB): Calculated for C₁₃H₁₄O₇N₂ (M+1) 311.2713, found m/z=311.0887.

Synthesis of D—Ala derivative (4). The product was obtained as a whitesolid in the matter described above.

H¹ NMR (400 MHz, DMSO): δ12.68 (bs, 1H), 9.02 (d, J=7.1 Hz, 1H), 8.31(d, J=8.7 Hz, 2H), 8.11 (d, J=8.7 Hz, 2H), 4.51-4.41 (m, 1H), 1.35 (d,J=7.3 Hz, 3H). C¹³ NMR (300 MHz, DMSO): δ174.7, 165.4, 149.4, 140.4,129.8, 124.4, 49.3, 17.6. CIMS (CH₄): M=238 calculated for C₁₀H₁₀O₅N₂.Found m/z=239 (M+1).

Synthesis of Peptide 4 a.

BnNHL—Lys(Boc)NH₂: A solution of 470 mg (1 mmols) FmocL—Lys(Boc)OH, 230mg (1.2 mmols) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), 165 μL (1.5 mmols) benzylamine and a catalyticamount of DMAP in 25 mL DCM, was stirred at r.t. under argon for 2 h.After dilution with 25 mL DCM, the solution was washed with 5 mLsolution HCl 1 M. Solvent was removed to give the benzylamide as a whitesolid. This was deprotected by stirring with 50 mL solutionDCM:piperidine at 4:1, at r.t. for 30 min. Separation on silica gelcolumn with DCM:MeOH at 15:1 to 7:1, gave the amine in 90% yield (300mg).

¹H NMR (400 MHz, CDCl₃): δ7.71-7.59 (bt, 1H), 7.38-7.27 (m, 5H),4.63-4.53 (bt, 1H), 4.48 (d, J=5.9 Hz, 2H), 3.45-3.37 (m, 1H), 3.18-3.04(m, 2H), 1.94-1.82 (m, 1H), 1.68-1.41 (m, 5H), 1.42 (s, 9H). CIMS (NH₃):M=335 calculated for C₁₈H₂₉O₃N₃. Found m/z=336 (M+1).

BnNHL—Lys(Boc)—D—ProNH: To the solution of amine were added 340 mg (1mmols) acid fluoride of FmocD—Pro and 350 μL (2 mmols) DIEA in 50 mLDCM, and stirred at r.t. under argon for 1 h. After solvent removal,purification was completed on silica gel column with DCM:acetone at 5:2.Fmoc deprotection was carried out as above. After separation on silicagel column with DCM:MeOH at 10:1 then 7:1 (1% TEA), the amine wasobtained in 85% yield over the two steps.

¹H NMR (300 MHz, CDCl₃): δ8.03 (d, J=8.3 Hz, 1H), 7.39-7.23 (m, 5H),6.91-6.82 (m, 1H), 4.67-4.59 (bt, 1H), 4.57-4.31 (m, 3H), 3.81-3.72 (m,1H), 3.17-3.04 (m, 2H), 3.02-2.74 (m, 2H), 2.18-1.41 (m, 10H), 1.42 (s,9H). CIMS (NH₃): M=432 calculated for C₂₃H₃₆O₄N₄. Found m/z=433 (M+1).

BnNHL—Lys(Boc)—D—Pro—L—Ser(tBut)NH₂: To the above amine in 50 mL DCMwere added 380 mg (1 mmols) acid fluoride of FmocL—Ser(tBut) and 350 μL(2 mmols) DIEA, and the resulting solution was stirred under argon for 1h. After solvent removal, the purification was achieved by flashchromatography with DCM:acetone at 2:1. After Fmoc deprotection andseparation on silica gel column with DCM:MeOH at 14:1, the amine wasobtained as 420 mg of white solid in 75% yield.

¹H NMR (300 MHz, CDCl₃): δ7.66-7.58 (bt, 1H), 7.39-7.23 (m, 5H), 6.82(d, J=8.3 Hz, 1H), 4.79-4.71 (bt, 1H), 4.71-4.27 (m, 4H), 3.88-3.71 (m,1H), 3.65-3.13 (m, 6H), 2.31-1.46 (m, 10H), 1.46 (s, 9H), 1.15 (s, 9H).CIMS (NH₃): M=575 calculated for C₃₀H₄₉O₆N₅. Found m/z=576 (M+1).

BnNHL—Lys(Boc)—D—Pro—L—Ser(tBut)CONMe₂: Capping of the serine wasaccomplished with excess dimethylcarbamyl chloride and DIEA, by stirringthe solution with a catalytic amount of DMAP under argon for 3 h at r.t.Purification on a silica gel column with DCM:acetone at 1:1, gave thepeptide in 85% yield.

¹H NMR (300 MHz, CDCl₃): δ7.46 (d, J=8.3 Hz, 1H), 7.34-7.24 (m, 5H),5.43-5.32 (bt, 1H), 5.12-5.02 (bt, 1H), 3.74-3.56 (m, 4H), 3.18-3.06 (m,2H), 2.84 (s, 6H), 2.38-1.45 (m, 10H), 1.46 (s, 9H), 1.17 (s, 9H). ¹³CNMR (300 MHz, CDCl₃): δ172.3, 139.1, 128.9, 127.9, 127.6, 79.5, 74.2,62.2, 61.1, 54.1, 47.8, 43.6, 40.4, 36.5, 30.9, 29.5, 29.3, 28.9, 27.8,24.6, 22.9. CIMS (NH₃): M=646 calculated for C₃₃H₅₄O₆N₇. Found m/z=647(M+1).

BnNHL—Lys—D—Pro—L—Ser(tBut)CONMe₂ (7): The Boc was selectively removedby stirring the peptide in DCM:TFA at 3:1 for 30 min at r.t. After theexcess TFA was eliminated, the peptide was purified by gel filtration onSephadex LH-20 with DMF.

¹H NMR (400 MHz, DMSO): δ8.28 (t, J=5.8 Hz, 1H), 7.82 (d, J=8.2 Hz, 1H),7.65 (bs, 3H), 7.32-7.18 (m, 5H), 6.22 (d, J=6.3 Hz, 1H), 4.42-4.12 (m,5H), 3.74-3.36 (m, 4H), 2.72 (s, 6H), 2.08-1.16 (m, 10H), 1.12 (s, 9H).LRMS (FAB): M=546 calculated for C₃₃H₄₆O₅N₆. Found m/z=547 (M+1).

BnNHL—Lys—D—Pro—L—SerCONMe₂ (4 a): To the Boc protected peptide in 10 mLDCM stirred under argon at 0° C., were added 1.3 equivalents of TiCl₄solution 1 M in DCM. The solution was stirred for 15 min, after which 5mL saturated aqueous NaHCO₃ solution were added. The organic layer wasremoved, and the water evaporated under high vacuum. To the solidobtained were added 25 mL MeOH and the solids were filtered off. Furtherpurification was accomplished by gel chromatography Sephadex LH-20 usingMeOH as eluent, to give the peptide as a white solid.

¹H NMR (400 MHz, DMSO): δ8.28 (bt, 1H), 7.74 (d, J=8.2 Hz, 1H),7.33-7.18 (m, 5H), 6.6 (bs, 1H), 6.24-6.14 (m, 1H), 5.02-4.90 (m, 2H),4.39-4.17 (m, 5H), 3.85-3.49 (m, 4H), 2.90-2.82 (m, 2H), 2.77 (s, 6H),2.08-1.22 (m, 10H). ¹³C NMR (300 MHz, DMSO): δ172.4, 172.2, 158.9,140.4, 129.1, 128.0, 127.7, 127.5, 63.1, 60.9, 55.8, 53.8, 47.7, 42.8,36.7, 31.8, 30.1, 24.8, 23.3. LRMS (FAB): M=490 calculated forC₂₄H₃₈O₅N₆. Found m/z=491 (M+1).

Synthesis of Control Peptides (6) and (8)

BnNHD—ProBoc: To 500 mg (2.32 mmols) of BocProOH in 15 mL of DCM wereadded 187 μL (2.32 mmols) pyridine and 324 μL (3.48 mmols) cyanuricfluoride. The resulting solution was stirred under argon at r.t. for 1h. After dilution with DCM to 150 mL, the organic layer was washed with2 mL water. Solvent was removed, and to this crude material 253 μL (2.3mmols) benzylamine and 800 μL (4.6 mmols) DIEA were added in 20 mL DCM.The solution was stirred at r.t. under argon for 1.5 h. Solvent wasremoved and after purification by silica gel column chromatography(DCM:acetone at 10:1), the peptide was obtained as 670 mg of white solidin 96% yield.

¹H NMR (400 MHz, CD₃OD): δ7.27-7.37 (m, 4H), 7.20-7.29 (m, 1H),4.43-4.46 (m, 1H), 4.27-4.31 (m, 1H), 4.13-4.22 (m, 1H), 3.50-3.58 (m,1H), 3.38-3.46 (m, 3H), 2.18-2.31 (m, 1H), 1.81-2.02 (m, 3H), 1.47 (s,3H), 1.33 (s, 6H). CIMS (NH₃): M=305 calculated for C₁₇H₂₄O₃N₂. Foundm/z=306 (M+1).

BnNHD—Pro—L—Ser(tBut)Fmoc: To 304 mg (1 mmol) of BnNH—L—ProBoc wereadded 100 mL DCM:TFA at 4:1, and the solution was stirred at r.t. for 1h. The excess TFA was removed, and to a solution of this crude materialin 25 mL DCM were added 400 mg (1.04 mmols) of the acid fluoride ofFmocL—Ser(OtBut) and 2 eq. of DIEA. After stirring at r.t. for 45 min.,the solvent was removed and the peptide was purified by columnchromatography (DCM:acetone at 9:1) to give 550 mg of a white foam in97% yield. CIMS (NH₃) M=569 calculated for C₃₄H₃₉O₅N₃. Found m/z=570(M+1).

BnNHD—Pro—L—Ser(tBut)NH₂: Deprotection of the Fmoc group was achieved bystirring the substrate in a solution of DCM:Piperidine at 4:1 for 30min. Solvent was removed, and purification of the crude by flashchromatography with DCM:MeOH at 8:1, gave 315 mg amine in 94% yield.CIMS (NH₃): M=347 calculated for C₁₉H₂₉O₃N₃. Found m/z=348 (M+1).

BnNHD—Pro—L—Ser(tBut)NHCONMe₂: To the solution of amine in 10 mL DCM,DIEA and dimethylcarbamyl chloride were added in excess over a period of5-7 h, until the TLC does not indicate the presence of the amine.Solvent was removed. Purification on a silica gel column withDCM:acetone at 1:1 gave 300 mg product in 80% yield.

¹H NMR (400 MHz, CDCl₃): δ7.98 (t, J=6.1 Hz, 1H), 7.28-7.15 (m, 5H),5.15 (d, J=6.1 Hz, 1H), 4.72-4.25 (m, 4H), 4.06-3.55 (m, 4H), 2.59 (s,6H), 2.35-1.92 (m, 4H), 1.17 (s, 9H). CIMS (NH₃): M=418 calculated forC₂₂H₃₄O₄N₄. Found m/z=419 (M+1).

BnNHD—Pro—L—Ser(OH)NHCONMe₂ (6): Deprotection of the t-Butyl group wasaccomplished by stirring the peptide with a solution of DCM:TFA at 4:1for 1 h at r.t. Solvent was removed, and after purification by silicagel column chromatography (DCM:acetone:MeOH at 5:5:1), the product wasobtained as 110 mg of oil in 42% yield (87% yield based on recoveredstarting material). Longer reaction time or more TFA led todecomposition of starting material. Further purification was done bysize-exclusion chromatography on Sephadex LH-20 with MeOH.Recrystallization from DCM/hexane gave 100 mg of a white solid.

¹H NMR (400 MHz, DMSO): δ8.24 (t, J=6.1 Hz, 1H), 7.35-7.17 (m, 5H), 6.38(d, J=6.1 Hz, 1H), 4.72-4.25 (m, 4H), 4.06-3.55 (m, 4H), 2.59 (s, 6H),2.35-1.92 (m, 4H), 1.17 (s, 9H). ¹³C NMR (300 MHz, CDCl₃): δ171.11,170.89, 157.37, 137.78, 128.04, 126.92, 126.75, 64.08, 59.94, 53.21,47.11, 42.84, 35.66, 28.36, 24.27. HRMS (FAB): calculated for C₁₈H₂₆O₄N₄(M+1) 363.2032, found 363.2019. IR (polyethylene card): 3307, 2918,2849, 1659, 1651, 1643, 1634, 1538, 1472, 1462, 1231, 1065.

BnNHL—LysNHAc (8): To 250 mg (0.74 mmols) of the benzylamide of L—Lys(δNHBoc) in 20 mL DCM were added 200 mL TEA, 200 mL Ac₂O and a catalyticamount of DMAP. After stirring the mixture at r.t. for 1 h, the solventwas removed and purification was performed on a silica gel column withDCM:acetone at 1:1. After standard deprotection of Boc and removal ofthe excess TFA, 10 mL saturated NaHCO₃ were added and the free amineextracted several times with DCM. Further purification was achieved on aSephadex LH-20 column with MeOH.

¹H NMR (300 MHz, DMSO): δ8.55 (t, J=6.0 Hz, 1H), 8.11 (d, J=8.0 Hz, 1H),7.32-7.18 (m, 5H), 4.28-4.12 (m, 3H), 2.52-2.42 (m, 2H), 1.82 (s, 3H),1.66-1.16 (m, 6H). ¹³C NMR (300 MHz, DMSO): δ173.4, 172.5, 138.9, 128.5,127.5, 127.2, 53.9, 43.0, 39.9, 31.6, 28.5, 23.0, 21.5. CIMS (CH₄):M=277 calculated for C₁₅H₂₃O₂N₃. Found m/z=278 (M+1).

To 0.5 mmols prolinol in 25 mL DCM were added 1.2 equivalents Bocprotected aminoacid, 1.2 equivalents EDC and a catalytic amount of DMAP(FIG. 12). After stirring the mixture at r.t. for 2 h, 100 mL of DCMwere added and the solution was washed with 5 mL HCl 1 N. Solvent wasremoved and the product purified on silica gel column with DCM:acetoneat 1:1 to give a colorless oil in yields higher than 90%.

SProC1NHBoc: ¹H NMR (400 MHz, CDCl₃): δ5.48 (bt, 1H), 4.57 (d, J=6.1 Hz,1H), 4.28-4.18 (m, 1H), 4.02-3.78 (m, 2H), 3.74-3.40 (m, 4H), 2.12-1.78(m, 3H), 1.67-1.62 (m, 1H), 1.47 (s, 9H). CIMS (CH₄): M=258 calculatedfor C₁₂H₂₂O₄N₂. Found m/z=259 (M+1).

SProC2NHBoc: ¹H NMR (400 MHz, CDCl₃): δ5.28 (bt, 1H), 4.98 (bd, 1H),4.28-4.18 (m, 1H), 3.74-3.54 (m, 2H), 3.51-3.42 (m, 4H), 2.54 (t, J=7.3Hz, 2H), 2.15-1.87 (m, 3H), 1.68-1.55 (m, 1H), 1.45 (s, 9H). CIMS (NH₃):M=272 calculated for C₁₃H₂₄O₄N₂. Found m/z=273 (M+1).

SProC3NHBoc: ¹H NMR (400 MHz, CDCl₃): δ4.96 (bd, 1H), 4.75 (bt, 1H),4.28-4.18 (m, 1H), 3.82-3.74 (m, 2H), 3.62-3.47 (m, 3H), 3.24-3.12 (m,2H), 2.42-2.34 (m, 2H), 2.10-1.71 (m, 3H) 1.68-1.58 (m, 1H), 1.48 (s,9H). ¹³C NMR (300 MHz, CDCl₃): δ174.0, 156.7, 79.6, 67.4, 61.4, 48.5,40.3, 32.4, 28.8, 28.6, 25.4, 24.8. CIMS (NH₃): M=286 calculated forC₁₄H₂₆O₄N₂. Found m/z=287 (M+1).

SProC4NHBoc: ¹H NMR (400 MHz, CDCl₃): δ5.11 (bd, 1H), 4.65 (bt, 1H),4.28-4.20 (m, 1H), 3.72-3.47 (m, 4H), 3.22-3.11 (m, 2H), 2.37 (t, 2H,J=7.3 Hz), 2.13-1.81 (m, 3H), 1.74-1.52 (m, 4H), 1.47 (s, 9H). ¹³C NMR(300 MHz, CDCl₃): δ174.6, 156.5, 79.5, 68.0, 61.6, 48.5, 40.5, 34.8,29.9, 28.8, 28.7, 24.8, 22.1. CIMS (NH₃): M=300 calculated forC₁₅H₂₈O₄N₂. Found m/z=301 (M+1).

SProC5NHBoc: ¹H NMR (400 MHz, CDCl₃): δ5.14 (d, J=6.0 Hz, 1H), 4.54 (bt,1H), 4.28-4.20 (m, 1H), 3.72-3.45 (m, 4H), 3.18-3.07 (m, 2H), 2.73 (t,J=7.3 Hz, 2H), 2.12-1.72 (m, 3H), 1.74-1.42 (m, 7H), 1.47 (s, 9H). ¹³CNMR (300 MHz, CDCl₃): δ174.7, 156.5, 79.4, 67.7, 61.5, 48.5, 40.7, 35.3,30.3, 28.8, 28.6, 26.9, 24.7. CIMS (NH₃): M=314 calculated forC₁₆H₃₀O₄N₂. Found m/z=315 (M+1).

SProC6NHBoc: H¹ NMR (400 MHz, CDCl₃): δ5.28 (d, J=6.0 Hz, 1H), 4.52 (bt,1H), 4.29-4.21 (m, 1H), 3.74-3.42 (m, 4H), 3.18-3.07 (m, 1H), 2.72 (t,J=7.3 Hz, 2H), 2.22-1.84 (m, 3H), 1.72-1.34 (m, 9H), 1.47 (s, 9H). ¹³CNMR (300 MHz, CDCl₃): δ174.7, 154.2, 68.2, 61.6, 48.5, 35.4, 30.3, 29.4,28.8, 28.7, 26.9, 25.0, 24.8. CIMS (NH₃): M=228 calculated forC₁₇H₃₂O₄N₂. Found m/z=229 (M+1).

Deprotection of Boc was achieved with TFA:DCM at 1:3 in 30 min. Afterthe excess TFA was removed on high vacuum, the salt was dissolved in 1mL water and added to a small Dowex 50W×4-400 ion exchange column, andeluted with a solution of NH₄OH 1 M. Further purification was achievedusing a C18 cartridge (Waters), eluting with water. The amine wasobtained in 80% overall, as a colorless oil.

SProC1: ¹H NMR (400 MHz, DMSO): δ3.96-3.78 (m, 1H), 3.53-3.15 (m, 6H),1.94-1.72 (m, 4H). ¹³C NMR (300 MHz, DMSO): δ172.4, 63.3, 61.9, 59.6,58.6, 46.2, 44.5, 28.7, 27.3, 24.2, 21.9. CIMS (NH₃): M=158 calculatedfor C₇H₁₄O₂N₂. Found m/z=159 (M+1).

SProC2: ¹H NMR (400 MHz, DMSO): δ4.01-3.85 (m, 1H), 3.52-3.08 (m, 6H),2.52-2.34 (m, 2H), 1.98-1.77 (m, 4H). ¹³C NMR (300 MHz, DMSO): δ171.2,63.3, 62.1, 59.4, 59.3, 47.7, 46.1, 28.6, 27.6, 24.3, 22.2. CIMS (NH₃):M=172 calculated for C₈H₁₆O₂N₂. Found m/z=173 (M+1).

SProC3: ¹H NMR (400 MHz, DMSO): δ3.95-3.87 (m, 1H), 3.54-3.20 (m, 5H),2.94 (bt, 2H), 2.94-2.72 (m, 2H) 1.96-1.52 (m, 6H). ¹³C NMR (300 MHz,DMSO): δ171.9, 63.3, 62.1, 59.3, 59.1, 47.6, 46.1, 32.3, 31.7, 28.6,27.6, 24.3, 22.2. CIMS (NH₃): M=186 calculated for C₉H₁₈O₂N₂. Foundm/z=187 (M+1).

SProC4: ¹H NMR (400 MHz, DMSO): δ3.95-3.85 (m, 1H), 3.49-3.09 (m, 4H),2.94-2.48 (m, 2H) 2.37-2.12 (m, 2H), 1.96-1.34 (m, 8H). ¹³C NMR (300MHz, DMSO): δ171.9, 63.3, 62.1, 59.4, 59.3, 47.6, 46.1, 42.1, 34.6,33.9, 32.3, 28.6, 27.5, 24.3, 22.9, 22.6, 22.1, 21.6. CIMS (NH₃): M=200calculated for C₁₀H₂₀O₂N₂. Found m/z=201 (M+1).

SProC5: ¹H NMR (400 MHz, DMSO): δ3.88-3.74 (m, 1H), 3.52-3.22 (m, 4H),2.91-2.52 (m, 2H), 2.39-2.19 (m, 2H) 1.95-1.74 (m, 4H), 1.54-1.22 (m,6H). ¹³C NMR (300 MHz, DMSO): δ171.9, 63.4, 62.2, 59.4, 59.3, 47.6,46.1, 34.8, 34.1, 28.6, 27.5, 26.9, 25.6, 24.9, 24.3, 22.1. HRMS (FAB):calculated for C₁₁H₂₂O₂N₂ 214.3079. Found 214.1619.

RProC5: ¹³C NMR (300 MHz, DMSO): δ171.8, 63.3, 62.1, 59.4, 59.3, 47.6,46.1, 34.6, 33.9, 29.0, 28.6, 27.5, 26.5, 25.3, 24.7, 24.3, 22.1. CIMS(NH₃): M=214 calculated for C₁₁H₂₂O₂N₂. Found m/z=215 (M+1).

SProC6: ¹H NMR (400 MHz, DMSO): δ4.01-3.70 (m, 1H), 3.52-3.18 (m, 4H),2.93-2.48 (m, 2H), 2.39-2.14 (m, 2H) 1.95-1.68 (m, 4H), 1.54-1.18 (m,8H). ¹³C NMR (300 MHz, DMSO): δ172.0, 63.3, 62.1, 59.4, 47.6, 46.1,34.8, 34.1, 29.5, 28.6, 27.5, 27.1, 25.8, 25.2, 24.3, 22.1. CIMS (NH₃):M=228 calculated for C₁₂H₂₄O₂N₂. Found m/z=229 (M+1).

Preparation of in vivo Study Samples

For the in vivo experiments, deprotection of the S,RProCnNHBocderivatives was performed by stirring for 1 h in 3 M HCl in EtOAc.Solvent was removed and the oils were extensively dried on high vacuum.100 mM stock solutions of each derivative were made in sterile PBS pH7.4 and the pH of the resulting solutions was adjusted with concentratedNaOH to 7.

FIG. 13. Step (a). To 1 mL (9.9 mmols) diamine in 100 mL DCM were added2.45 g (9.9 mmols) BocON and 1.4 mL TEA, and the solution was stirred atr.t. overnight. The product was purified on silica gel column withDCM:MeOH at 1:1 (5% TEA) to give 1.8 g of white solid in 96% yield.

¹H NMR (400 MHz, CDCl₃): δ4.68 (bt, 1H), 3.18 (bq, 2H), 2.72 (t, J=6.7Hz, 2H), 1.60-1.50 (m, 2H), 1.52 (s, 9H), 1.31-1.21 (m, 2H).

FIG. 13. Step (b). To 83 mg (0.5 mmols) of L—ProOMe.HCl were added 100μL TEA followed by 1 mL solution 2 M of phosgene in toluene, and themixture was stirred at r.t. for 1 h. After a careful removal of excessphosgene, the product was dissolved in 5 mL DCM and added to a solutionof 140 μL (1.3 mmols) pyridine and 94 mg (0.5 mmols) amine in 15 mL DCM.The solution was stirred at r.t. for 2 h. Solvent removal, followed bypurification on silica gel column with DCM:acetone at 1:1 gave 150 mg ofproduct as a colorless oil, in 90% yield.

¹H NMR (400 MHz, CDCl₃): δ4.67-4.58 (m, 1H), 4.57-4.51 (m, 1H),4.47-4.41 (m, 1H), 3.74 (s, 3H), 3.52-3.22 (m, 4H), 3.19-3.08 (m, 2H),2.17-1.97 (m, 4H), 1.60-1.50 (m, 4H), 1.48 (s, 9H). CIMS (NH₃): M=343calculated for C₁₆H₂₉O₅N₃. Found m/z=344 (M+1).

FIG. 13. Step (c). To the methyl ester were added 166 mg (10 eq.) NaBH₄in 15 mL MeOH and the solution was stirred at r.t. for 20 min and thenrefluxed for 2 h. Purification on silica gel column with DCM:acetone at1:1 gave the product quantitatively as a colorless oil.

¹H NMR (400 MHz, CDCl₃): δ5.18 (bd, 1H), 4.86 (bd, 1H), 4.65 (bt, 1H),4.17-4.05 (m, 1H), 3.72-3.52 (m, 2H), 3.40-3.11 (m, 6H), 2.11-1.87 (m,2H), 1.62-1.57 (m, 4H), 1.48 (s, 9H). ¹³C NMR (300 MHz, CDCl₃): δ159.6,156.7, 79.5, 68.0, 60.6, 47.2, 40.7, 40.6, 29.0, 28.8, 27.8, 27.7, 24.5.CIMS (NH₃): M=315 calculated for C₁₅H₂₉O₄N₃. Found m/z=316 (M+1).

FIG. 13. Step (d). Deprotection of the Boc group and work up of theresulting amine, were completed as described for ProCn.

¹H NMR (400 MHz, DMSO): δ6.37 (bt, 1H), 3.71-3.63 (m, 1H), 3.44-3.18 (m,4H), 3.12-2.90 (m, 2H), 2.73 (bt, 2H), 1.88-1.68 (m, 4H), 1.55-1.35 (m,2H). ¹³C NMR (300 MHz, DMSO): δ158.4, 64.5, 59.5, 47.0, 28.4, 27.9,24.1. CIMS (CH₄): M=215 calculated for C₁₀H₂₁O₂N₃. Found m/z=216 (M+1).

In vitro Hydrolysis of D—Ala—D—Lac

The ability of peptide 4 a in cleaving the substrate 5 was assessed inaqueous phosphate buffer pH 7 at 37° C. Using HPLC and monitoring thep-NO₂-phenyl derivative by UV at 275 nm, we could easily follow thedisappearance of D—Ala—D—Lac derivative 5 and the formation of thehydrolyzed product 4. No significant effect on the rate of hydrolysisover buffer was observed using the control sequences 6, 7, 8 alone or 6and 8 combined (FIG. 14). A 20% hydrolysis of D—Ala—D—Lac in thepresence of 4 a was observed after 24 h.

The prolinol derivatives were additionally, tested for their ability tocleave 5 in aqueous phosphate buffer pH 7.0 at 37° C. A 50% D—Ala—D—Lachydrolysis was observed after 24 h implying that the SProC5 derivativeis twice as active as the initial peptide 4 a. Activity declines in theSProCn series with the decrease of the chain length from 5 carbons to 1carbon. This result can be explained not only by a decrease inefficiency of the terminal amino in reaching to the carboxylate andester of D—Ala—D—Lac with the shortening of the chain, but also by thedecrease in nucleophilicity of the hydroxyl. A 6 carbon chain is alsoless active. The lower activity of SProC6 is probably due to the higherflexibility of the carbon chain that does not render the amino groupavailable for the reaction.

To confirm these speculations we compared the chemical shifts of the OHand NH in the H¹ NMR spectra of the NHBoc protected SProCn series (Table7). The study shows that there is a competition between the amino groupand the hydroxyl for hydrogen bonding to the amide. With the decrease ofthe chain length, the probability of the OH being hydrogen bondeddecreases substantially, fact reflected in the chemical shifts of the OHand NH with the change in the length of the carbon chain.

TABLE 7 Shifts in the NMR position of OH and NHBoc (Boc- protected smallmolecule series) with the modification of the carbon chain length. Boc-OH position NH position derivative (ppm) (ppm) SProC1 4.57 5.48 SProC24.98 5.28 SProC3 4.96 4.75 SProC4 5.11 4.65 SProC5 5.14 4.54 SProC6 5.284.52 SProUC4 4.86 4.65

The table also explains the low activity of SProUC4, derivative in whichthe amide is replaced by urea. The urea is a better acceptor than theamide and should increase the reactivity of the hydroxyl. However, it ispossible that structural constraints imposed by the urea play animportant role and do not allow for proper orientation for H-bonding.

Mechanistic Studies

To get insights on the mechanism of this reaction, the cleavage of 1 by4 a was studied in THF-5% water. Stock solutions of 2 mM concentrationof 1 in THF and 49 mM of 4 a (lyophilized from PIPES buffer pH 7.0) inwater were prepared. In three ampoules were added 40 μL solution of 1, 7μL of 4 a and the volume was adjusted to 160 μL with THF. For backgroundmeasurements, in another three ampoules 7 μL water were added instead of4 a to the solution of 1. All six ampoules were sealed under an argonstream and placed in an oil bath heated at 60° C. For each measurementone vial was opened and 5 μL were taken, diluted with 5 μL THF and 2 μLof this solution were injected in the HPLC.

The reaction proceeds with the formation of a transesterificationproduct 3, which then is cleaved by water (Scheme 3).

The reaction could be monitored by HPLC at 485 nm, and the separation ofthe three components was easily performed on an analytical reverse phasecolumn using a gradient of acetonitrile:water. Isolation of theintermediate 3 proved to be however, more difficult. Application of theassay mixture to a size exclusion column (Sephadex LH-20 with DMF) gavea fraction enriched in 3, and this was used for a COSY-¹H NMR analysis(FIG. 17). Comparison of the NMR spectra of 4 a, 3 and 9 (FIG. 16)confirmed the identity of the intermediate 3. Mass spectrum analysis(MS) was used to additionally establish the identity of the three peaksseen in the HPLC chromatogram (FIG. 15). The above description is forthe purposes of teaching the person of ordinary skill in the art how topractice the invention, and is not intended to detail all those obviousmodifications and variations of it, which will become apparent to theskilled worker upon reading the description. It is intended, however,that all such obvious modifications and variations be included withinthe scope of the present invention, which is defined by the followingclaims.

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What is claimed is:
 1. A method of treating a subject afflicted with aninfection caused by vancomycin resistant Gram-positive bacteria inwhich, resistance results from the conversion of an amide bond to anester bond in the cell wall peptide precursors of the bacteria whichcomprises administering to the subject an antibacterial amount ofvancomycin or a homolog of vancomycin and an amount of an agenteffective to selectively cleave said ester bond so as to thereby treatthe subject.
 2. The method of claim 1, wherein the subject is a humanbeing.
 3. The method of claim 1, wherein the agent is an activatednucleophile, is not a peptide, and is further characterized by thepresence within the agent of an electrophile and chirality complementaryto a bacterial cell wall peptide.
 4. The method of claim 1, where theagent catalytically cleaves said ester bond.
 5. The method of claim 1,wherein said ester bond is present in the structure D—Ala—D—Lac.
 6. Themethod of claim 1, wherein the agent is administered prior toadministering vancomycin or the homolog of vancomycin.
 7. The method ofclaim 4, wherein the agent is administered a sufficient period of timeprior to administering vancomycin or the homolog of vancomycin to permitcleavage of said ester bond to be effected.
 8. The method of claim 1,wherein the agent and vancomycin or the homolog of vancomycin areadministered simultaneously.
 9. The method of claim 8, wherein the agentis covalently attached to vancomycin or the homolog of vancomycin. 10.The method of claim 1, wherein the bacteria are Van A, Van B, Van D orVan G Gram positive bacteria.
 11. The method of claim 1, wherein thebacteria are Staphylococcus bacteria.
 12. The method of claim 10,wherein the bacteria are S. aureus bacteria.
 13. The method of claim 1,wherein the bacteria are Enterococcus bacteria.
 14. The method of claim1, wherein the bacteria are Streptococcus bacteria.
 15. The method ofclaim 1, wherein the bacteria are Leuconostoc bacteria.
 16. The methodof claim 1, wherein the bacteria are Pediococcus bacteria.
 17. Themethod of claim 1, wherein the bacteria are Lactobacillus bacteria. 18.The method of claim 1, wherein the bacteria are Erysipelothrix bacteria.19. A method of killing vancomycin resistant Van A, Van B, Van D, or VanG Gram-positive bacteria which comprises contacting the bacteria with anagent that selectively cleaves D—Ala—D—Lac cell wall depsipeptide in thebacteria in an amount effective to cleave such depsipeptide and anantibacterial amount of vancomycin or a homolog of vancomycin so as tothereby kill the bacteria.
 20. The method of claim 19, wherein the agentis an activated nucleophile, is not a peptide, and is furthercharacterized by the presence within the agent of an electrophile andchirality complementary to a bacterial cell wall depsipeptide.
 21. Themethod of claim 19, where the agent catalytically cleaves saidD—Ala—D—Lac cell wall depsipeptide.
 22. The method of claim 19, whereinthe agent is administered prior to administering vancomycin or thehomolog of vancomycin.
 23. The method of claim 22, wherein the agent isadministered a sufficient period of time prior to administeringvancomycin or the homolog of vancomycin to permit cleavage of theD—Ala—D—Lac cell wall depsipeptide to be effected.
 24. The method ofclaim 19, wherein the agent and vancomycin or the homolog of vancomycinare administered simultaneously.
 25. The method of claim 24, wherein theagent is covalently attached to vancomycin or the homolog of vancomycin.26. The method of claim 19, wherein the bacteria are Staphylocoecusbacteria.
 27. The method of claim 26, wherein the bacteria are S. aureusbacteria.
 28. The method of claim 19, wherein the bacteria areEnterococcus bacteria.
 29. The method of claim 19, wherein the bacteriaare Streptococcus bacteria.
 30. A method of treating a subject afflictedwith an infection caused by glycopeptide antibiotic resistantGram-positive bacteria in which resistance results from the conversionof an amide bond to an ester bond in the cell wall peptide precursors ofthe bacteria which comprises administering to the subject anantibacterial amount of a glycopeptide antibiotic and an amount of anagent effective to selectively cleave said ester bond so as to therebytreat the subject.
 31. A method of killing glycopeptide antibioticresistant Gram-positive bacteria which comprises contacting the bacteriawith an agent that selectively cleaves D—Ala—D—Lac cell walldepsipeptide in the bacteria in an amount effective to cleave suchdepsipeptide and an antibacterial amount of the glycopeptide antibioticso as to thereby kill the bacteria.
 32. The method of claim 31, whereinthe bacteria are Staphylococcus bacteria.
 33. The method of claim 31,wherein the bacteria are S. aureus bacteria.
 34. The method of claim 31,wherein the bacteria are Enterococcus bacteria.
 35. The method of claim31, wherein the bacteria are Streptococcus bacteria.
 36. The method ofclaim 31, wherein the bacteria are Leuconostoc bacteria.
 37. The methodof claim 31, wherein the bacteria are Pediococcus bacteria.
 38. Themethod of claim 31, wherein the bacteria are Lactobacillus bacteria. 39.The method of claim 31, wherein the bacteria are Erysipelothrixbacteria.
 40. The method of claim 19, wherein the bacteria areLeuconostoc bacteria.
 41. The method of claim 19, wherein the bacteriaare Pediococcus bacteria.
 42. The method of claim 19, wherein thebacteria are Lactobacillus bacteria.
 43. The method of claim 19, whereinthe bacteria are Erysipelothrix bacteria.
 44. The method of claim 30,wherein the subject is a human being.
 45. The method of claim 30,wherein the agent is an activated nucleophile, is not a peptide, and isfurther characterized by the presence within the agent of anelectrophile and chirality complementary to a bacterial cell wallpeptide.
 46. The method of claim 30, wherein the agent has thestructure:

wherein n is an integer from 1 to 6 inclusive and R is hydrogen or a C₁to C₆ straight chain or branched alkyl group.
 47. The method of claim30, where the agent catalytically cleaves said ester bond.
 48. Themethod of claim 30, wherein said ester bond is present in the structureD—Ala—D—Lac.
 49. The method of claim 30, wherein the agent isadministered prior to administering the glycopeptide antibiotic.
 50. Themethod of claim 49, wherein the agent is administered a sufficientperiod of time prior to administering the glycopeptide antibiotic topermit cleavage of said ester bond to be effected.
 51. The method ofclaim 30, wherein the agent and the glycopeptide antibiotic areadministered simultaneously.
 52. The method of claim 51, wherein theagent is covalently attached to the glycopeptide antibiotic.
 53. Themethod of claim 30, wherein the bacteria are Staphylococcus bacteria.54. The method of claim 53, wherein the bacteria are S. aureus bacteria.55. The method of claim 30, wherein the bacteria are Enterococcusbacteria.
 56. The method of claim 30, wherein the bacteria areStreptococcus bacteria.
 57. The method of claim 30, wherein the bacteriaare Leuconostoc bacteria.
 58. The method of claim 30, wherein thebacteria are Pediococcus bacteria.
 59. The method of claim 30, whereinthe bacteria are Lactobacillus bacteria.
 60. The method of claim 30,wherein the bacteria are Erysipelothrix bacteria.
 61. The method ofclaim 31, wherein the agent is an activated nucleophile, is not apeptide, and is further characterized by the presence within the agentof an electrophile and chirality complementary to a bacterial cell walldepsipeptide.
 62. The method of claim 31, wherein the agent has thestructure:

wherein n is an integer from 1 to 6 inclusive and R is hydrogen or a C₁to C₆ straight chain or branched alkyl group.
 63. The method of claim31, where the agent catalytically cleaves the D—Ala—D—Lac cell walldepsipeptide.
 64. The method of claim 31, wherein the agent isadministered prior to administering the glycopeptide antibiotic.
 65. Themethod of claim 31, wherein the agent is administered a sufficientperiod of time prior to administering the glycopeptide antibiotic topermit cleavage of the D—Ala—D—Lac depsipeptide to be effected.
 66. Themethod of claim 31, wherein the agent and the glycopeptide antibioticare administered simultaneously.
 67. The method of claim 66, wherein theagent is covalently attached to the glycopeptide antibiotic.
 68. Themethod of claim 1, wherein the agent has the structure:

wherein n is an integer from 1 to 6 inclusive and R is hydrogen or a C₁to C₆ straight chain or branched alkyl group.
 69. The method of claim19, wherein the agent has the structure:

wherein n is an integer from 1 to 6 inclusive and R is hydrogen or a C₁to C₆ straight chain or branched alkyl group.
 70. The method of claim46, wherein n=5 and R═H.
 71. The method of claim 62, wherein n=5 andR═H.
 72. The method of claim 68, wherein n=5 and R═H.
 73. The method ofclaim 69, wherein n=5 and R═H.